A versatile "pan-spike-in" internal reference genome methodology for quantitative ChIP-seq normalization

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Project summary: ChIP-seq has enabled the mapping of genomic occupancy of many chromatin factors and epigenetic histone modifications, linked to normal growth and development and to the pathogenesis of many human diseases. ChIP-seq can also measure changes in the occupancy levels of epigenomic marks/factors between different chromatin samples such as control vs drug-treated cells. However, accurate quantitation in ChIP-seq requires effective and reliable sample normalization. ChIP with reference exogenous genome or ChIP- Rx devised by Orlando et al (2014) provides a rigorous normalization approach via addition of crosslinked Drosophila S2 cells (reference exogenous chromatin) with crosslinked human cells on a per-cell basis. Antibody cross-reactivity to the exogenous and experimental test chromatin is critical for the success of this spike-in approach. Exogenous reference species Drosophila, C. glabrata and S. pombe do not share many of the epigenomic/chromatin factors with the experimental human/mouse or S. cerevisiae models. Moreover, lack of “ChIP grade” antibody has led to epitope tagging of chromatin factors. To overcome the antibody cross-reactivity barriers, we hypothesized that a chromatin-binding protein in the exogenous reference genome when fused to the IgG-binding domains of Protein A and G (PAG tag) will cross-react with antibodies raised in rabbit or mouse recognizing epigenomic/chromatin factors or epitope-tags, and thus can serve as an ‘one-for-all or pan spike-in’ for normalization. Based on successfully using a C. glabrata ‘pan spike-in’ exogenous reference for normalization and measurement of chromatin occupancy differences for epigenome-modulating factors in S. cerevisiae, we propose to now create a Drosophila S2R+ ‘pan spike-in’ reference genome for ChIP-Rx in human or mouse cells. We propose to also create S. pombe ‘pan spike-in’ reference genome as a non-pathogenic alternative to C. glabrata, and a ‘pan spike-in’ S. cerevisiae reference for ChIP-Rx in S. pombe, all in Aim 1. We will validate the utility of the created ‘pan-spike-in’ genomes for normalization by measuring the subunit-dependent chromatin occupancy of Set1 H3K4 methyltransferase from yeast to humans using ChIP-Rx (Aim 2). In Aim 3, we propose to diversify the ‘pan-spike-in’ normalization system by testing other immunoglobulin-binding domains to capture goat or chicken IgG. Overall, successful completion of the proposed studies will lead to the creation of valuable reagents for not only epigenomics/chromatin researchers but also to the broader research community, by providing a robust and accurate normalization methodology for ChIP-seq to quantify epigenome differences among cell populations, treatments and genomic states. The studies resulting from using the ‘pan spike-in’ system generated here will yield novel mechanistic insights and also enable accurate quantitation of global and local chromatin modifications that is needed for the discovery and characterization of epigenome regulators and for the drugs targeting them in a variety of human diseases ranging from developmental, metabolic, neurological disorders to cancers.